How are some genes that specify cell fates regulated by elements in their 3' untranslated region (3'UTR) so that cells adopt a given fate at the right time and place in development? We will investigate this fundamental question of biology by analyzing how the 3'UTR of the C. elegans sex determination gene tra-2 controls development. Two regulatory processes act via the 3'UTR. The first is crucial for hermaphrodite development, and controls translation of the tra-2 mRNA by two 3'UTR elements (called TGEs). GLD-l is a TGE dependent translational repressor, and the gene laf-1 is also required for control. The second regulation is essential for development of both hermaphrodites and males, and controls the activity of the zinc finger transcription factor, TRA-1. TRA-1 binding to the tra-2 3'UTR inhibits TRA-1 transcriptional regulatory activity by promoting the nuclear export of the protein. The export of tra-2 mRNA and TRA-1 are mutually dependent on one another suggesting they exit the nucleus as a complex. In the next five years, we propose to investigate how the tra-2 3'UTR controls development. First using transgenic and biochemical assays, we will investigate the molecular mechanism by which TGEs and GLD-1 inhibit translation. Recent evidence suggests that GLD-1 inhibits translation via the Poly(A) Binding Protein (PAB). We will generate mutations in GLD-1 that disrupts the GLD-1/PAB interaction, and ask if these proteins fail to repress translation. In addition, we will ask if TRA-1 also affects TGE dependent control. Second, we will investigate if laf-1 is a regulatory RNA, as molecular analysis suggests the gene encodes no significant open reading frames. Also, we will ask when and where laf-1 gene product is expressed. Third, we will investigate how regulated nuclear export of tra-2 mRNA and TRA-1 is accomplished. Preliminary data indicates CeNXF-2, a protein similar to Mex67/TAP, is necessary for tra-2 mRNA nuclear retention. Using transgenes and RNA gel shift, we will confirm and extend these findings. In addition, we will identify the sequences of TRA-1 and the tra-2 3'UTR required for TRA-1 RNA binding and export. Finally, we will investigate the role of export in sex determination by asking if any known sex determining genes control TRA-1/tra-2 mRNA nuclear export. The health relatedness of this work derives from its contribution to an understanding of fundamental control of gene activity by 3'UTRs. The TGE control is present in several metazoans, including man, and governs the translation of the human oncogene GLI1, suggesting this control plays a role in suppressing tumorigenesis.
